1. Field of the Invention
The present invention relates to a process for the separation of poly-.beta.-hydroxyalkanoic acid (PHA) of high molecular weight from a microbial, PHA-producing biomass, by treatment with hypochlorite either alone or in combination with a surfactant.
2. Background of the Art
Poly-.beta.-hydroxyalkanoic acid (PHA) is an optically active linear polyester made of various small .beta.-hydroxymonocarboxylic acids. It can be produced synthetically but is more cheaply produced by microorganisms. Many micro-organisms accumulate large quantities of PHA intracellularly when their growth is limited by some element other than carbon. The excess carbon is converted into PHA as a mechanism of carbon and energy storage. The form of PHA most commonly produced by microorganisms is a homopolymer of .beta.-hydroxybutyric acid. This homopolymer is called poly-.beta.-hydroxybutyric acid (PHB). Many PHB-accumulating microorganisms will produce PHA copolymers if certain organic acids such as propionic acid are added to their medium during the PHA accumulation phase.
Since PHA is an intracellular product, one of the limitations to the economic feasibility of its production is the cost of separating the PHA from the rest of the microbial biomass. Many methods have been developed to achieve this but they all have serious drawbacks.
A number of solvent extraction processes have been developed to separate PHA from its native biomass. These usually involve the use of chlorinated solvents such as chloroform (U.S. Pat. No. 4,324,907 and U.S. Pat. No. 4,358,583), dichloroethane (U.S. Pat. No. 4,391,766 and U.S. Pat. No. 4,324,907) or 1,1,2-trichloroethane (U.S. Pat. No. 4,310,684). Propylene or ethylene carbonate (U.S. Pat. No. 4,101,533) has also been used. Apart from being expensive, these processes generally involve working with large quantities of toxic and/or explosive volatile solvents. Moreover, in most of the cases, the PHA that has been extracted, must be subsequently precipitated by addition of the extracting solution to an alcohol such as methanol. In the case of propylene carbonate, PHB is soluble up to 340 g/L at 150.degree. C. but to only 3 g/L at 100.degree. C. Therefore cooling is used to obtain the purified polymer. In practice cells are often spray or freeze-dried before extraction. They may also be washed with acetone to remove polar lipids and excess water which may interfere with the extraction process.
European laid-open patent application (EP-A 0,145,233) discloses an expensive and complicated method comprising a thermal treatment of the PHA containing-biomass, followed by an enzymatic treatment and then a washing with an anionic surfactant to dissolve the cellular, non-PHA biomass.
A less complex method is also disclosed by Williamson and Wilkinson (J. Gen. Microbiol. 19:198-203 (1958)), in which the biomass is subjected to differential digestion by the sodium hypochlorite solution. Although simple and effective, this method however has been avoided up to now because it has been reported to cause severe degradation of the PHA molecular weight (Lundgren et al, J. Bacteriol. 89:245-251 (1965); Nuti et al, Can. J. Microbiol. 18:1257-1261 (1972); Dawes and Senior, Adv. Microbiol. Physiol. 10:135-266 (1973)). As a matter of fact the highest molecular weight which has been reported to have been obtained by this method was 22,000 (Lundgren et al, J. Bacteriol.89:245-251 (1965)).